Live attenuated vaccines can mimic natural infection and induce humoral and cellular immune response. However, the possibility of reassortment between vaccine viruses and field isolates and of mutations from low-pathogenic to highly pathogenic viruses has prevented the use of live attenuated strains as poultry vaccines. In ovo vaccination using live attenuated strains that can undergo limited replication cycles would be a better option, because these strains can be used for mass vaccination without spreading or reassorting with other viruses. Our previous study demonstrated that two influenza nonstructural (NS) variant viruses are highly attenuated and immunogenic in chickens, making them potential live vaccine candidates. In this study, we tested whether NS variants could be used as in ovo vaccines alone or in combination with temperature-sensitive (ts) mutations. In addition, we also tested the effect of different hemagglutinin (HA) subtypes on in ovo vaccination of NS variants. Our results demonstrated that NS variants alone or in combination with ts mutations were not attenuated enough to be used for in ovo vaccination. We also observed variable effects of different HA subtypes in the same NS deletion variant backbone on hatchability. However, even with substitution of HA subtypes, NS variant–inoculated eggs still had lower hatchability compared to the mock control group, indicating that the high virulence of NS variant backbone strain in eggs might have affected the results.